microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C Product Description The microrna cdna Synthesis Mix has been designed to easily convert micrornas into cdna templates for qpcr starting from total RNA or RNA preparations pre-enriched for micrornas. However, recent studies have shown that Total RNA preparations and pre-enrichment for micrornas is no longer necessary. MicroRNAs are not naturally polyadenylated. With the microrna cdna Synthesis Kit, micrornas are polyadenylated through a poly(a) polymerase reaction. Then, ReadyScript Reverse Transcriptase and other necessary reagents for cdna synthesis are subsequently added to convert the poly(a) tailed micrornas into cdna using an oligo-dt adapter primer. The adapter primer incorporates a unique sequence at its 5 end which allows for amplification of cdnas in real-time RT-qPCR reactions. The kit includes a Human Positive Control Primer that can be used to quantify a target gene that is ubiquitously expressed in most human tissues, the small nucleolar RNA SNORD44. There is sufficient Poly(A) Tailing Buffer and microrna cdna Reaction Mix to accommodate the use of no poly(a) polymerase and no reverse transcriptase control reactions. Individual micrornas are quantified in real-time SYBR Green RT-qPCR reactions with the specific microrna qpcr Assay Primer and the Universal PCR Primer (which binds specifically to the unique sequence incorporated into the cdna by the oligo-dt adapter primer during the RT reaction). The pre-designed and validated microrna Assays provide maximum sensitivity and specificity in RT-qPCR amplification and quantification of micrornas. s Provided Description Poly (A) Tailing Buffer (5X) Human Positive Control Primer Nuclease-free Water Universal PCR Primer Catalog No. MIRRT01 MIRRT02 MIRRT03 MIRRT04 25 RXN 60 25 1500 125 Poly (A) Polymerase MIRRT05 25 microrna cdna Reaction Mix ReadyScript Reverse Transcriptase MIRRT06 MIRRT07 s Required, but not Provided 270 25 RXN 240 1500 500 1080 Description Catalog No. Vendor Universal PCR Primer (as necessary) MIRUP- 500RXN microrna qpcr Assay Primer and/or various Control primer microrna SYBR Green qpcr ReadyMix Instrument dependent Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. Storage/Stability microrna cdna Synthesis Mix is stable for 1 year when stored in a constant temperature freezer at -20 C
MicroRNA analysis using the microrna Quantification System. With the microrna cdna Synthesis Mix, micrornas are first polyadenylated in a poly(a) polymerase reaction (Step 1). ReadyScript Reverse Transcriptase and other necessary reagents for cdna synthesis are subsequently added to convert the poly(a) tailed micrornas into first-strand cdna using an oligo-dt adapter primer (Step 2). The unique sequence at the 5 end of the adapter primer allows amplification of microrna cdnas in real-time SYBR Green RT-qPCR reactions using the microrna qpcr Universal Primer and individual microrna assays (Step 3). Step 1. Polyadenylation of micrornas Poly (A) Tailing Buffer (5X) RNA (up to 1 μg total RNA or micrornaenriched RNA) Nuclease-free water Poly (A) Polymerase Total 2 Up to 7 1 10 3. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect the components at the bottom of the reaction tube. 4. Incubate: 60 minutes at 37 C 5 minutes at 70 C 5. Briefly centrifuge to collect the contents. Keep on ice before cdna synthesis. If using ng or less of total RNA, the incubation at 37 C can be shortened to 20 minutes Step 2. First-strand cdna synthesis First-strand cdna Synthesis Reaction Set up the cdna Synthesis Reaction: Poly (A) Tailing Reaction (from final step above) microrna cdna Reaction Mix ReadyScript Reverse Transcriptase Total 10 9.0 1.0 20 Step 3. Real-Time SYBR Green RT-qPCR amplification of micrornas 6. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect the components. 7. Incubate 20 minutes at 42 C* 5 minutes at 85 C *The RT reaction can be run at 45 C to reduce background without affecting the sensitivity. Procedure Poly(A) Tailing Reaction 1. After thawing components (except enzyme), place components on ice. Mix, and then briefly centrifuge to collect contents at the bottom of the tube. 2. Combine the following reagents in 0.2-mL microtubes or a 96-well plate sitting on ice. The cdna is ready for RT-qPCR. If needed, the microrna cdna product can be diluted with water or 10 mm Tris-HCl (ph 8.0), 0.1 mm EDTA (recommended for long-term storage). Diluted cdna is stable for several months at 4 C. The cdna can be stored long term at -20 C. 2
Procedure Real Time SYBR Green RT-qPCR Amplification of MicroRNAs Real-time SYBR Green RT-qPCR is performed using 200 nm of each microrna qpcr Assay Primer and Universal PCR Primer along with the appropriate microrna SYBR Green qpcr ReadyMix product depending on the instrument platform being used. Please refer to the instrument reference table to select the proper formula for your instrument. For each RT-qPCR reaction add the following components: microrna SYBR Green qpcr ReadyMix (2X) microrna qpcr Assay Primer (10 μm) Universal PCR Primer (10 μm) microrna cdna (0.1 10 ng) Nuclease-free water Total 25 1.0 1.0 50 The amount of microrna cdna can be adjusted depending on the expression level of the microrna. As a starting point use about 1 ng of total RNA equivalent per RT-qPCR reaction. For micrornas expressed at low levels you may use 10 ng of total RNA equivalent per RT-qPCR reaction. For most applications 20 to 25 µl RT-qPCR reaction volumes are suitable but reaction volumes can be scaled up or down as needed. 3-Step Cycling Procedure (optional) Pre-incubation / activation 95 C for 2 minutes PCR (40 cycles) Denature 95 C for 5 seconds Anneal 60 C for 15 seconds Extend 70 C for 15 seconds (collect fluorescence data) Procedure Real Time SYBR Green RT-qPCR Amplification of MicroRNAs (continued) Use of a slightly higher annealing temperature (63 C) in the 2-step Cycling Procedure may improve the specificity of some assays. Melt curve analysis is optional. Most microrna RT-qPCR reactions will produce a single, slightly broader first-derivative melt peak compared to reactions using two gene-specific primers due to slight heterogeneity in the poly(a) tail length. Quality Control Kit components are free of contaminating DNase and RNase. ReadyScript Reverse Transcriptase is functionally tested in reverse transcription quantitative PCR (RT-qPCR). First-strand synthesis is performed in triplicate on each dilution of a log-fold serial dilution of HeLa cell total RNA from 1 pg to 1 μg. One-tenth of each first-strand reaction is used for qpcr amplification. Kinetic analysis must demonstrate linear resolution over five orders of dynamic range (r2 > 0.995) and a PCR efficiency > 90%. 2-Step Cycling Procedure (recommended) Pre-incubation / activation 95 C for 2 minutes PCR (40 cycles) Denature 95 C for 5 seconds Anneal 60 C for 30 seconds (collect fluorescence data) 3
Real-time PCR Instrument Name microrna SYBR Green qpcr ReadyMix microrna SYBR Green qpcr ReadyMix Low ROX microrna SYBR Green qpcr ReadyMix with ROX microrna SYBR Green qpcr ReadyMix iq MIRRM00 MIRRM01 MIRRM02 MIRRM03 Applied Biosystems 5700 Applied Biosystems 7000 Applied Biosystems 7300 Applied Biosystems 7500 Applied Biosystems 7500 Fast Biosystems 7700Applied Applied Biosystems 7900 Applied Biosystems 7900 HT Fast Applied Biosystems 7900HT Applied Biosystems StepOnePlus Applied Biosystems StepOne Applied Biosystems ViiA 7 Bio-Rad CFX384 Bio-Rad CFX96 Bio-Rad icycler iq Bio-Rad iq 5 Bio-Rad MiniOpticon Bio-Rad MyiQ Bio-Rad/MJ Chromo4 Bio-Rad/MJ Opticon 2 Bio-Rad/MJ Opticon Cepheid SmartCycler Eppendorf Mastercycler ep realplex Eppendorf Mastercycler ep realplex2 s Illumina Eco qpcr Qiagen/Corbett Rotor-Gene 3000 Qiagen/Corbett Rotor-Gene 6000 Qiagen/Corbett Rotor-Gene Q Roche LightCycler 480 Stratagene Mx3000P Stratagene Mx3005P Stratagene Mx4000 4
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Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 The purchase of this product includes a limited, nontransferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. Applicable only to products containing Passive Reference The purchase of this product includes a limited, non-transferable right to use the purchased amount of the product to perform Applied Biosystems patented Passive Reference Method for the purchaser s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. For information about these rights or on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. SYBR is a registered trademark of Molecular Probes, Inc is a trademark of Quanta Biosciences, ReadyMix and ReadyScript are trademarks of Co. LLC. LightCycler is a registered Trademark of Roche. Applied Biosystems, StepOne, StepOnePlus, ViiA, and ROX are trademarks Life Technologies Corporation. Stratagene, MX3000P, MX3005P and MX4000 are trademarks of Agilent Technologies, Inc. Mastercycler is a trademark of Eppendorf. Rotor-Gene is a registered trademark of Qiagen GmbH. SmartCycler is a trademark of Cepheid. CFX96, CFX384, icycler iq, iq5, MyiQ, Opticon, MiniOpticon and Chromo4 are trademarks of Bio-Rad Laboratories SC,ER,PHC 06/12-1 2012 Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Co. LLC, registered in the US and other countries. Sigma brand products are sold through, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip. 5